Universidade de Aveiro 2018
Departamento de Biologia
Pedro Araújo Carvalho Impacto da idade em proteínas de sinalização de
espermatozoides
DECLARAÇÃO
Declaro que este relatório é integralmente da minha autoria, estando
devidamente referenciadas as fontes e obras consultadas, bem como
identificadas de modo claro as citações dessas obras. Não contém, por isso,
qualquer tipo de plágio quer de textos publicados, qualquer que seja o meio dessa
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Universidade de Aveiro 2018
Departamento de Biologia
Pedro Araújo Carvalho
Impacto da idade em proteínas de sinalização de
espermatozoides
Impact of age on spermatozoa signaling proteins
Dissertação apresentada à Universidade de Aveiro para cumprimento dos requisitos necessários à obtenção do grau de Mestre em Biologia Molecular e Celular, realizada sob a orientação científica da Doutora Margarida Sâncio da Cruz Fardilha, Professora Auxiliar do Departamento das Ciências Médicas da Universidade de Aveiro e co-orientação da Doutora Joana Vieira Silva, Investigadora do Instituto de Investigação e Inovação em Saúde.
o júri
presidente Professor Doutor Mário Guilherme Garcês Pacheco
Professor Auxiliar com Agregação do Departamento de Biologia da Universidade de Aveiro
vogal – arguente principal
vogal - co-orientadora
Doutora Maria João Martinho de Freitas
Investigadora de Pós-Doutoramento do Departamento de Ciências Médicas da Universidade de Aveiro
Doutora Joana Vieira Silva
Investigadora de Pós-Doutoramento do Departamento de Ciências Médicas da Universidade de Aveiro
orientadora Professora Doutora Margarida Sâncio da Cruz Fardilha
Professora Auxiliar do Departamento de Ciências Médicas da Universidade de Aveiro
Agradecimentos À Professora Doutora Margarida Fardilha, por todo os ensinamentos que me transmitiu ao longo do meu percurso académico. Destaco e agradeço a sua franqueza, humildade e disponibilidade.
À Doutora Joana Vieira Silva, pela enorme ajuda e total disponibilidade que demonstrou bem como todos os ensinamentos que me passou de forma coerente. Foi uma das pessoas mais altruístas e humildes que conheci neste último ano académico e sem ela, esta dissertação não era possível.
À Dra. Madalena Cabral, pois sem ela não teria tido a oportunidade de estagiar na COGE e de proceder à recolha das amostras para a realização deste trabalho. Aprendi com ela que é possível ser-se profissional, ambicioso e curioso. É um exemplo para mim pois demonstrou-me a paixão que tem por este tema que tanto me fascina.
À Dra. Carla Leal, pelo companheirismo e boa-disposição. Por ser uma das pessoas mais divertidas que conheci em ambiente laboratorial. Agradeço também os ensinamentos que me transmitiu.
Ao Miguel, pela ajuda na análise estatística.
A todos os meus amigos, em particular à Luísa e à Diana, por estarem sempre presentes nos momentos mais difíceis e nelas encontrar sempre uma palavra amiga de força e encorajamento.
A toda as minhas colegas de trabalho e alunos do Cubo Mágico pela paciência e persistência.
Por fim e em especial, aos meus pais, Augusto e Virgínia, e aos meus irmãos, André e Andreia, pela motivação e por sempre acreditarem em mim. Agradeço as palavras de consolação e por sempre me motivarem a prosseguir mais além e por acreditarem na minha paixão.
palavras-chave (in)fertilidade masculina, espermatozoide, transdução de sinal, PRAS40, P70 S6 quinase.
resumo A infertilidade pode ser definida como a incapacidade de conceber uma gravidez após pelo menos doze meses de relações sexuais regulares desprotegidas. O fator masculino encontra-se envolvido em aproximadamente 50% dos casos de infertilidade conjugal, sendo exclusivamente responsável em aproximadamente 20% dos casos. O adiamento da paternidade sugere que a idade pode ser uma causa de problemas reprodutivos. Embora esteja bem documentado que as mulheres apresentam um declínio na fecundidade com o avançar da idade, os dados disponíveis sobre os efeitos do envelhecimento na fertilidade masculina mostram uma maior disparidade. Apesar de ser cientificamente consensual que a idade masculina é um fator importante, muito pouco se sabe sobre os mecanismos moleculares subjacentes à relação entre idade masculina e fertilidade reduzida.
O objetivo deste estudo foi avaliar o impacto do envelhecimento em espermatozoides humanos. Para esse fim, os parâmetros seminais básicos e os níveis de 18 proteínas de sinalização foram analisados em 31 homens que recorreram a Técnicas de Procriação Medicamente Assistida (PMA).
O presente estudo revelou que a idade do sexo masculino estava associada à percentagem de defeitos da peça intermediária e à presença de citoplasma residual em excesso (CRE) em espermatozoides.
Este estudo revelou ainda que o nível de duas fosfoproteínas de espermatozoides humanos, PRAS40 (Thr246) e P70 S6 quinase (Thr389) apresentaram uma correlação significativamente negativa (p<0,01) com idade masculina. Assim, pôde-se concluir que essas duas fosfoproteínas podem ser consideradas bons marcadores para a monotorização do declínio da fertilidade masculina intrínseca ao processo de envelhecimento. Contudo, mais estudos com um maior número de amostras deverão ser realizados.
keywords male (in)fertility, spermatozoa, signal transduction, PRAS40, P70 S6 kinase.
Abstract Infertility is defined as the inability to achieve a pregnancy after twelve or more months of unprotected regular intercourse. The male factor is involved in approximately 50% of the cases of conjugal infertility, being exclusively responsible in approximately 20% of the cases. The postponement of paternity suggests that age may be a cause of reproductive problems. While it is well documented that women have a decline in fecundity with age, the data available regarding the effects of age on male fertility show a wider disparity. Despite the scientific consensus that male age is an important factor, very little is known about the molecular mechanisms underlying the connections between male age and reduced fertility.
The aim of this study was to evaluate the impact of aging on human spermatozoa. To that end, the basic seminal parameters and the levels of 18 signaling proteins were analyzed in 31 men who resort to Assisted Reproductive Techniques (ART).
The present study revealed that male age was associated with the percentage of midpiece defects and the presence of excess residual cytoplasm (ERC) in spermatozoa.
This study also showed that the level of two phosphoproteins in human spermatozoa, PRAS40 (Thr246) and P70 S6 kinase (Thr389), presented a significant negative correlation (p<0.01) with male age. Therefore, these two phosphoproteins may be good markers to monitor the male fertility decline intrinsic to the aging process. Although more and larger studies must be conducted.
Table of contents
List of abbreviations ... 1
List of figures ... 4
List of tables ... 4
1. General Introduction and Objective
... 41.1. Testis ... 5 1.1.1. Leydig cells ... 5 1.1.2. Sertoli cells ... 6 1.2. Spermatozoa ... 7 1.2.1. Spermatogenesis ... 7 1.2.2. Spermatozoa structure ... 8 1.3. Epididymis ... 10
1.3.1. Epidydimal sperm maturation ... 11
1.4. Sperm capacitation ... 12
1.5. Signaling pathways in spermatozoa ... 14
1.5.1. sAC/cAMP/PKA ... 14
1.5.2. Phospholipase C ... 15
1.5.3. ROS/MAPK pathway ... 17
1.6. Infertility ... 19
1.6.1. Impact of age on male infertility ... 19
1.7. Objective ... 21
2. Material and Methods
... 222.1. Study Overview ... 22
2.2. Human samples collection ... 22
2.3. Basic Semen Analysis ... 22
2.3.1. Macroscopy parameters evaluation ... 22
2.3.2. Microscopy parameters evaluation ... 23
2.3.3. Semen Processing – Density gradients ... 24
2.4. Semen Cryopreservation ... 25
2.5. Spermatozoa Protein Extracts ... 25
2.6. Protein Quantification - Bicinchoninic Acid Assay ... 25
2.7. Antibody Array - PathScan® Intracellular Signaling Array ... 26
2.8. Statistical analysis ... 26
3. Results
... 274. Discussion ... 35
5. References ... 40
6. Appendix ... 50
6.1. Appendix 1 ... 50 6.2. Appendix 2: ... 51University of Aveiro
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List of abbreviations
AC Adenylyl cyclase
AI Artificial Insemination
AKAPs A-kinase anchoring proteins
AMP Adenosine monophosphate
ART Assisted Reproduction Technology
ATP Adenosine-5-triphosphate
BCA Bicinchoninic acid assay
BSA Bovine serum albumin
Ca2+ Calcium
cAMP Cyclic adenosine monophosphate
CatSper channel Cation channels of sperm
CD45 Leukocyte common antigen
CDK16 Cyclin-dependent kinase 16
COGE Clínica Obstétrica e Ginecológica de Espinho
DAG Diacylglycerol
DNA Deoxyribonucleic acid
FS Fibrous sheet
FSH Follicle-stimulating hormone
G6PDH Glucose-6-phosphate dehydrogenase
GH Growth hormone
Glu Glutamic acid
GPR18 G protein-coupled receptor 18
GPX5 Type 5 glutathione peroxidase
GSK3 Glycogen synthase kinase 3
H2O2 Hydrogen peroxide
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HCO3- Bicarbonate
iBiMED Institute for Research in Biomedicine
ICSI Intracytoplasmic sperm injection
IVF In vitro fertilization
IZUMO1 Sperm-egg fusion protein 1
LH Luteinizing hormone
MAPK/ERK Mitogen-activated protein kinase
MS Mitochondrial sheath
mTOR Mammalian target of rapamycin
NADPH Nicotinamide adenine dinucleotide phosphate
NO- Nitric oxide
O2- Superoxide
ODFs Outers dense fibers
P70 S6 Kinase Ribosomal protein S6 kinase beta-1
PDE Phosphodiesterase
pH Potencial of Hydrogen
PI3 Phosphoinositol 3-phosphate
PIP2 Phosphotidylinositol 4,5-biphosphate
PKA Protein kinase A
PKB/AKT Protein kinase B
PKC Protein kinase C
PLC Phospholipase C
PPP1 Phosphoprotein phosphatase 1
PRAS40 Proline-rich AKT1 substrate 1
Raf Rapidly accelerated fibrosarcoma
ROS Reactive oxygen species
RPS6 Ribossomal protein S6
sAC soluble adenylyl cyclase
Ser Serine
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Shc Src homology collagen
SOC Store-operated channels
Thr Threonine
tRNA Transfer ribonucleic acid
Tyr Tyrosine
WHO World Health Organization
WR Working Reagent
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List of figures
Figure 1 - Testicles and schematic cross-section of a testicular tubule………6
Figure 2 - Schematic illustration of spermatogenesis……….8
Figure 3 - Schematic representation of the spermatozoa………....10
Figure 4 - Schematic representation of sAC/cAMP/PKA pathway in sperm………15
Figure 5 - Schematic representation of Phospholipase C pathway in sperm………16
Figure 6 - Schematic representation of ROS/MAPK pathway in sperm………18
Figure 7 - Scatter plots about correlation between age and the signaling proteins PRAS40 and p70 S6 kinase.………34
Figure 8 - Schematic representation of PI3K/AKT1/mTORC1/P70-S6K/RPS6KB1 and it's possible effect……….39
List of tables
Table 1 - Standards for BCA assay……….26Table 2 - Basic semen parameters of 31 patients providing semen samples for ART treatments or sperm analysis………28-29 Table 3 - Expression patterns of 18 well-characterized signaling molecules when phosphorylated or cleaved (PathScan® Intracellular Signaling Array) ……….30-31 Table 4 - Associations between patients age and the results obtained from the basic seminal analyses………32 Table 5 - Associations between age and the results obtained from the expression patterns of 18 well-characterized signaling molecules when phosphorylated or cleaved (PathScan® Intracellular Signaling Array) ……….………33-34
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1.
General Introduction and Objective
The male reproductive system consists of two testicles (male gonads), a system of genital ducts, the accessory glands (prostate, bulbourethral gland and seminal vesicles) and the penis. The testicles produce male gametes (spermatozoa) through spermatogenesis and secrete male sex hormones, testosterone.
1.1. Testis
The testicles are the male gonads, responsible for the synthesis of male germ cells as well as sex hormones. They are oval structures involved by the scrotum and suspended by the spermatic cord (Figure 1). The testicles are surrounded by the tunica albuginea, that contains abundant contractile elements (Middendorff et al., 2002). The testicular parenchyma is composed of seminiferous tubules constituting the testicular pulp, where the production of spermatozoa occurs, and is supported by loose connective tissue (Kerr, 1992).
1.1.1. Leydig cells
Cells with endocrine function are found adjacent to the blood vessels, the Leydig cells. They are responsible for the production of the male sex hormone, testosterone. There are two generations of Leydig cells – fetal and adult (Prince, 2001). The fetal generation of Leydig cells (from birth to the first year of age) resulting from Chorionic Gonadotropin (hCG) stimulation have round nucleus, abundant smooth endoplasmic reticulum and mitochondria with tubular cristae (Nistal et al., 1986). The adult generation, under the stimulation of the Luteinizing Hormone (LH) has the role of producing testosterone, from puberty and during the entire life (Benton, Shan and Hardy, 1995). Human Leydig cells contain large Reinke crystalloids of variable size and number (Kerr, 1991). The high levels of circulating testosterone at puberty cause the testis cords to canalize and become the seminiferous tubules (Clermont and Huckins, 1961).
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1.1.2. Sertoli cells
From the basal lamina to the lumen of the seminiferous tubule, there are Sertoli cells involved in mechanical support and nutrition of germ cells providing critical factors necessary for the successful progression of germ cells into spermatozoa, through the complex process of spermatogenesis (Griswold, 1998; Kerr, 1992; Silva and Carvalho, 2010) (see section 1.2.1.). Sertoli cells have receptors to testosterone and Follicle-stimulating hormone (FSH) (McLachlan et al., 2006). Testosterone has a negative feedback on the pituitary gland suppressing LH secretion (Damassa et al., 1976). Sertoli cells are also responsible to the maintenance of the integrity of the seminiferous epithelium by establishing tight junctions (blood-testis barrier) (Tsukita, Furuse and Itoh, 2001; Pelletier and Byers, 1992).
Figure 1 - Testicles (A) Cross-section showing the location of the seminiferous tubules, the vas
deferens and the epididymis. Adapted from Desai et al., 2017. (B) Schematic cross-section of a testicular tubule, illustrating Sertoli cells, peritubular myoid cells and Leydig cells (in the interstitium). Retrieved from Cooke and Saunders, 2002.
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1.2. Spermatozoa
Germ cells divide and differentiate forming the male gametes (spermatozoa), though spermatogenesis (Griswold, 1998; Kerr, 1992; Silva and Carvalho, 2010).
1.2.1. Spermatogenesis
In humans, the progression from spermatogonia to spermatozoa take approximately 64 days (Heller and Clermont, 1963). In the seminiferous tubules, we can find cells increasing the order of maturation: spermatogonia, spermatocytes (primary and secondary), spermatids and spermatozoa (Figure 2). Spermatogenesis is a process that occurs from the periphery to the lumen of the tubules and requires several hormones namely LH, FSH, testosterone and growth hormone (GH) (Sadler, 2011). In humans, spermatogenesis is divided into three different phases: mitotic phase, meiotic phase (I and II) and spermiogenesis (Kanakis and Goulis, 2015).
Approximately at the same time, primordial germ cells give rise to spermatogonia, diploid cells that can be of two types: spermatogonia type A, which divide by mitosis to form part of a continuous reserve of stem cells and spermatogonia of type B, which by mitosis give rise to primary spermatocytes, during approximately 22 days, followed by rapid termination of meiosis I and formation of secondary spermatocytes (Bras et al., 1996). During the second meiotic division, these cells begin to form haploid spermatids. The last stage of spermatogenesis, spermiogenesis, constitutes the spermatozoa formation from spermatids. Includes, for instance, the formation of the acrosome, which covers half of the nuclear surface and contains enzymes that help fertilization (Zaneveld and De Jonge, 2013) and when fully formed, spermatozoa are carried by the contractile elements present in the walls of the seminiferous tubules and the tubular fluid secreted by the Sertoli cells into the epididymis, where sperm motility is acquired (Haschek, Rousseaux and Wallig, 2009).
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Figure 2 – Schematic illustration of spermatogenesis. Retrieved from Rato et al., 2012.
1.2.2. Spermatozoa structure
The spermatozoa may be divided into two regions: the head and the flagellum (Figure 3). The head is about 4,39 µm long (Bellastella et al., 2010) and compromises the nucleus, protected by the perinuclear theca (Terada et al., 2000), which contains the DNA (Deoxyribonucleic acid) condensing core and protamines, positively charged DNA proteins rich in arginines (Balhorn, 2007), responsible to enable nuclear hypercondensation and ensure the sperm genome remains inactive until it can be deposited inside an egg and reactivated (Brewer, Corzett and Balhorn, 2002). There are two types of protamines: protamine 1 (P1) and the family of protamine 2 (P2, P3 and P4); genes protamine mutations and changes in their expression are associated with male infertility (Oliva, 2006).
The acrosome is a Golgi-derived secretory vesicle that covers 47.5% of the head (Bellastella et al., 2010). This structure containing hydrolytic enzymes (as acrosin and hyaluronidase) that digest zona pellucida, helping the sperm penetrate the oocyte (Yoshinaga and Toshimori, 2003). Measuring acrosin activity is a suitable approach for
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evaluating the fertilizing capacity of human spermatozoa, because men with leukospermia (presence of leukocytes in the ejaculate above the threshold value (WHO, 2010)), and abnormal semen parameters present low actin activity (Zalata et al., 2004). The hyaluronidase is responsible to enable acrosome-intact sperm to reach the zona pellucida by hyaluronan hydrolysis (Kimura et al., 2009).
The flagellum consists of four regions: connecting piece, mid-piece, principal piece and final piece (Inaba, 2003). The connecting piece contains a proximal centriole and distal centriole that gives rise to the axoneme, which extends throughout the length of the entire flagellum requiring proteins such as tubulin, nexin, and dynein for its formation (Jin, Wang and Fang, 2014). The mid-piece presents nine outers dense fibers (ODFs) and a mitochondrial sheath (MS) that encloses the ODFs and the axoneme (Olson and Sammons, 1980). Mitochondria are found only in the MS of the mid-piece. As in other cells, sperm mitochondria produce Adenosine-5-triphosphate (ATP) through aerobic respiration. The ODFs extend into the principal piece of the flagellum (Turner, 2005; Inaba, 2003). Between the end of the mid-piece and the beginning of the principal piece are the annulus (Turner, 2005). Into the principal piece, the MS terminates and two of the ODFs are replaced by two longitudinal columns of fibrous sheet (FS). The FS is restricted to the principal piece and has kinase anchoring proteins (AKAPs) like A-kinase anchor protein 4 (AKAP4). AKAPs tether cyclic AMP-dependent protein A-kinases and thereby localize phosphorylation of target proteins and initiation of signal-transduction processes triggered by cAMP (Cyclic adenosine monophosphate) (Miki et al., 2002; Lindemann and Lesich, 2010). The final piece is the short terminal portion of the flagellum and has only the axoneme surrounded by the plasma membrane (Turner, 2005).
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Figure 3 - Schematic representation of the spermatozoa. The head contains the acrosome and the
nucleus. The flagellum is divided structurally into four areas: the connecting piece, mid-piece, principal piece and end-piece. Retrieved from Fardilha, Silva and Conde, 2015.
1.3. Epididymis
Although the human epididymis does not have defined sections in comparison to other primates (Cooper, 2012), this segmented organ can be divided into four main anatomical regions: initial segment (closest to the testis), caput (region between the initial segment and the corpus), corpus and cauda (closest with the vas deferens) (Martan, 1969; Ivell, 2007). The segments display distinct ions concentrations and differential expression of genes, essential to regulate sperm maturation. Spermatozoa need to undergo two
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maturational processes and successive biochemical changes to become competent to fertilize the oocyte: maturation in the epididymis (male reproductive system) and capacitation (female reproductive system) (Bearer and Friend, 1990).
1.3.1. Epidydimal sperm maturation
After spermiation (release of immature sperm into the lumen of the seminiferous tubules), immature spermatozoa need to undergo a maturation process in the epididymis to become motile. Those include alterations in intracellular pH, calcium
(Ca2+) concentration and cAMP responsible for proteins phosphorylation (Huang and
Vijayaraghavan, 2004). Spermatozoa are subject to morphological and biochemical changes such as post-translational protein modifications, cytoplasmic droplet (cytoplasmic excess) migration along the middle piece and a decrease of the head size (Fardilha, Silva and Conde, 2015; Cooper, 2011). As well, during epidydimal transit spermatozoa acquire new proteins like zona pellucida sperm-binding protein (ZP3), enzymes of the polyol pathway (HE5/CD52), type 5 glutathione peroxidase (GPX5) and sperm adhesion molecule 1 (PH-20) (Sullivan, Frenette and Girouard, 2007). These proteins are responsible for sperm maturation and they are present in the epididymosomes, membranous vesicles secreted in the intraluminal compartment of the epididymis (Saez, Frenette and Sullivan, 2003).
During the epididymal transit, the levels of Ca2+ decrease and the levels of intracellular
pH and cAMP increase. It is known that cAMP is responsible for the activation of protein kinase A (PKA) and this leads the initiation and stimulation of spermatozoa motility (Chakrabarti et al., 2007).
Ser/Thr phosphatases play a key role in activation of sperm motility (Tash and Bracho, 1994; Ickowicz, Finkelstein and Breitbart, 2012). For instance, phosphoprotein phosphatase 1 catalytic subunit gamma 2 (PPP1CC2), a testis-enriched and sperm-specific PPP1 isoform, is a key player in sperm motility acquisition (Fardilha et al., 2011; Fardilha et al., 2013). In caput region, spermatozoa are immotile. Glycogen synthase kinase 3 (GSK3) phosphorylates PPP1R2 at Thr73 which inhibits the interaction between PPP1R2 and PPP1CC2 resulting in active PPP1CC2 (Silva, Freitas and Fardilha, 2014). Also,
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spermatozoa at this stage have high cholesterol, non-phosphorylated sperm-egg fusion protein 1 (IZUMO1), low membrane fluidity and low levels of transfer ribonucleic acid (tRNA) fragments (Calvel, et al., 2010; Leahy and Gadella, 2015; Ellerman et al., 2010). Contrary, in the cauda region, PPP2CC2 is associated to PPP1R2 and consequently
inactive (Somanath, Jack and Vijayaraghavan, 2004). Ca2+ influx activates sAC, which
produces cAMP activating Rap guanine nucleotide exchange factor (RAPGEFs), which activates Protein kinase B (PKB), also know AKT, that phosphorylates GSK3. GSK3 inhibition leads to decrease Thr73 PPP1R2 phosphorylation. Consequently, PPP1R2 binds PPP1CC2. As well, a multimeric complex has been identified composed by PPP1CC2, sds22 (PPP1R7), actin and I3 (PPP1R11), where PPP1CC2 was inactive. Thus, PPP1 activity is inhibited and ser/thr phosphorylation of key residues increases leading to motile spermatozoa (Korrodi-Gregório et al., 2013). Cauda spermatozoa have high phospholipid content, extensively phosphorylated IZUMO1, high membrane fluidity and high levels of tRNA fragments (Calvel et al., 2010; Miranda et al., 2009; Leahy and Gadella, 2015). Mature spermatozoa are stored in the cauda region of the epididymis (Fardilha, Silva and Conde, 2015). Other functions like spermatozoa immunoprotection also occur in the epididymis (Marchiani et al., 2017).
1.4. Sperm capacitation
Sperm capacitation is a physiological process that spermatozoa must undergo to acquire fertilization capability, being necessary structural and biochemical changes that occur in the spermatozoa during passage through the female reproductive tract (López-González et al., 2014). Spermatozoa is a differentiated cell devoid of transcription and translation machinery and, after ejaculation, these cells are deposited in the vagina, an environment with hostile conditions like acidic internal pH (Qi et al., 2007) and a high concentration of progesterone and albumin (Abou-haila and Tulsiani, 2009). Progesterone is a messenger responsible for hyperactivation of the motility,
cross-reaction and chemotaxis of sperm with the effect of Ca2+ induction (Lishko, Botchkina
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After passage through the cervix, spermatozoa arrive at the uterus undergoing several structural and biochemical changes, such as the loss of plasma membrane cholesterol (Martínez and Morros, 1996). After this, proteins secreted by oviductal tissue have been associated with capacity-related molecular events, such as tyrosine (Tyr) phosphorylation of sperm proteins and the ability to respond to acrosome reaction (Zumoffen et al., 2010). Also, these proteins bind to the spermatozoa and protect them from oxidative damages induced by reactive oxygen species (ROS) and stimulate sperm motility (Huang et al., 2013). For instance, sperm fucosyltransferase-5 (sFUT5) is a membrane carbohydrate-binding protein on human spermatozoa involved in spermatozoa–oviduct interaction that contributes to the success of fertilization (Huang et al., 2015). A recent study, showed that peroxiredoxins (antioxidant enzymes) are necessary to control the concentration of ROS generated during capacitation and infertile men have lower levels of peroxiredoxins (Lee et al., 2017). Other process occurs during capacitation is the acquisition of hyper-activated motility that results of phospholipase D-dependent actin polymerization, allowing sperm to penetrate with greater flexibility the mucus present in the fallopian tubes (Itach et al., 2012). As well, ionic alterations occur during sperm capacitation: the increasing of intracellular
bicarbonate (HCO3-) and Ca2+ concentration, through voltage-dependent channels and
cation channels of sperm (CatSper), leads to the activation of soluble adenylyl cyclase (sAC), which in turn promotes the increase of cAMP activating PKA leading to the
production of superoxide (O2.-), causing the increase of phosphorylated proteins (Chung
et al., 2017; Cruz et al., 2014; Qi et al., 2007; Fardilha, Silva and Conde, 2015; Alasmari
et al., 2013). HCO3- influx also leads to the increasing of intracellular pH, causing of the
sperm plasma membrane hyperpolarization (López-González et al., 2014; Leemans et al., 2016).
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1.5. Signaling pathways in spermatozoa
1.5.1. sAC/cAMP/PKA
The sAC/cAMP/PKA pathway is responsible for protein phosphorylation in the spermatozoa (Abou-haila and Tulsiani, 2009). cAMP has been reported to be essential for events occurring during capacitation, including activation of motility, hyperactivation and acrosome reaction (Buffone et al., 2014).
sAC plays a critical role in cAMP signaling in spermatozoa: the increase of HCO3- and Ca2+
activate the sAC, which is responsible to convert AMP in cAMP (Xie et al., 2006). PKA is not essential for spermatogenesis and spermiogenesis, however, PKA does play a critical role in sperm capacitation and motility that are required for fertilization (Burton and McKnight, 2007). PKA structure consists of two regulatory subunits (RIα and RIIα) and two catalytic subunits (Cα1 and Cα2). RIα is expressed throughout spermatogenesis, while RIIα only appears at the late stages in spermatogenesis (Burton and McKnight, 2007). The increase of cAMP leads to the activation of PKA which causes the dissociation of active sperm catalytic subunit (Cα2). Cα2 phosphorylates downstream targets that
lead sperm capacitation by increasing motility, Ca2+ entry and tyr phosphorylation
(Burton and McKnight, 2007; Kaupp and Strünker, 2017). PKA inhibits the production of cAMP by directly or indirectly inhibiting sAC activity. PKA activation also leads to an increase in actin polymerization, a process responsible for the development of hyperactivated motility (Ickowicz, Finkelstein and Breitbart, 2012). The process of activation/inhibition of this pathway is obtained by negative feedback, the action of phosphodiesterase (PDE) and activation of specific ser/tyr phosphatases (Fardilha, Silva
and Conde, 2015). With the increase of the Ca2+ in the intracellular fluid, this ion binds
to the calmodulin, multifunctional Ca2+ binding protein, forming a complex that is
responsible for the activation of PDE. PDE hydrolyses cAMP in AMP, inhibiting
sAC/cAMP/PKA pathway. Thereby, Ca2+ has a dual function: it is a secondary messenger
in the activation of sAC and participates in regulation when binding to calmodulin, inhibiting sAC/cAMP/PKA pathway.
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Figure 4 - Schematic representation of sAC/cAMP/PKA pathway in sperm. The activation of this
pathway is obtained by the increase of HCO3- and Ca2+. These ions activate the sAC, which is
responsible to convert AMP in cAMP. The increase of cAMP leads to the activation of PKA which causes the dissociation of Cα2, leading sperm capacitation. The inhibition of this pathway is obtained by sAC inhibition by PKA complex and the increase of calmodulin/Ca2+ complex that is responsible
for the activation of PDE. PDE hydrolyses cAMP in AMP. sAC: soluble adenylyl cyclase; AMP: adenosine monophosphate; cAMP: cyclic adenosine monophosphate; PKA: protein kinase A; Cα2: active sperm catalytic subunit; PDE: phosphodiesterase. Adapted from Fardilha, Silva and Conde, 2015; Buffone et al., 2014.
1.5.2. Phospholipase C
Phospholipase C/PI3/Ca2+ is signal pathway essential for actin dispersion and acrosome
reaction (Breitbart, 2003). This pathway is initiated when progesterone and ZP3 bound to receptors localized in the anterior region of the spermatozoa head: inhibitory G protein-coupled receptors (Gi) and tyr kinase receptors (Schwartz et al., 2016).
Phospholipase C isoforms include six families of enzymes (PLC-β, γ, δ, ε, ζ and η), based on their biochemical proprieties, that play a key role in a wide array of intracellular signaling pathways (Béziau et al., 2015). PLC-β is associated to G protein-coupled receptors (McCudden et al., 2005). In turn, PLC-γ is associated with tyr kinase receptors
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(Nishibe et al., 1990; Nakamura and Fukami, 2017). These two isoforms of the phospholipase C hydrolyse phosphotidylinositol 4,5-biphosphate (PIP2) to phosphoinositol 3-phosphate (PI3) and diacylglycerol (DAG) (Breitbart, 2003). PI3 induce
the release of Ca2+ from internal Ca2+store (acrosome) to the cytosol (Walensky and
Snyder, 1995). DAG activates the protein kinase C (PKC) to open Ca2+channels in the
plasma membrane (Breitbart, 2002; Fukami et al., 2010). Progesterone also induces
Ca2+ influx into spermatozoa, essential for sperm hyperactivation, acrosome reaction and chemotaxis towards the egg (Lishko, Botchkina and Kirichok, 2011).
The PIP2 hydrolysis, the depletion of Ca2+ in the acrosome and the increase of Ca2+ concentration in the cytosol leads to actin hydrolysis (Breitbart and Finkelstein, 2015). The polymerization of the actin allows the approach of the acrossomatic and plasmatic membranes and their fusion, which is essential for the acrosome reaction (Fardilha, Silva and Conde, 2015). The Ca2+ depletion in the acrosomic zone stimulates the action of SOC allowing the Ca2+ influx of the extracellular medium to the cytosol (Breitbart, 2002).
Figure 5 - Schematic representation of Phospholipase C pathway in sperm. Progesterone and ZP3
bound to Gi and tyr kinase receptors, which are associated PLC-β1 and PLC-γ, respectively. PLC isoforms hydrolyse PIP2 to DAG and PI3. DAG activates PKC that activates Ca2+ channels. PI3 induces
Ca2+ release from acrosome to the cytosol, leading actin hydrolysis and consequently acrosome
reaction. ZP3: zona pellucida sperm-binding protein 3; Gi: inhibitory G protein-coupled receptors; tyr: tyrosine; PLC-β1: phospholipase C β1 isoform; PLC-γ: phospholipase C γ isoform; PIP2: phosphotidylinositol 4,5-biphosphate; DAG: diacylglycerol; PI3: phosphoinositol 3-phosphate. PKC: protein kinase C. Adapted from Fardilha, Silva and Conde, 2015; Breitbart, 2003.
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1.5.3. ROS/MAPK pathway
ROS such as O2-, hydrogen peroxide (H
2O2) and nitric oxide (NO-) may have contradictory
effects: high levels of ROS, mostly H2O2, have deleterious effects on spermatozoa and
may cause irreversible damage in sperm DNA (de Lamirande and Gagnon, 1995). However, in low and controlled levels, ROS are essential for spermatozoa capacitation (De Lamirande, Leclerc and Gagnon, 1997) modulating signals pathways, like mitogen-activated protein kinase (MAPK) pathway (Bailey, 2010).
The signal transduction is initiated by Shc (Src homology collagen) which will be phosphorylated and after this, Shc active Grb2 (Growth factor receptor-bound protein 2). Shc and Grb2 are examples of adapter proteins (Fardilha, Silva and Conde, 2015). Grb2 activate Sos (son of sevenless) and Sos activate Ras (family of GTP-binding proteins), responsible to activate ERK cascade that it plays a role upstream of protein tyr
phosphorylation (O'flaherty, de Lamirande and Gagnon, 2006). H2O2, a major ROS in
sperm, activates protein kinase C, which will activate rapidly accelerated fibrosarcoma
(Raf). PI3K phosphorylates PDK1 which phosphorylates Akt. Akt stimulates NO
-synthetase resulting in increased concentration of this ion which consequently activates Ras (Aitken, 2017; Fardilha, Silva and Conde, 2015). Components of the extracellular signal-regulated kinase (ERK) family of MAPK pathways are present in spermatozoa (Urnerand and Sakkas, 2003). Raf phosphorylates MEK, specific kinases for the thre-glu-tyre module, present in ser/thr-specific kinases, like ERK1 and ERK2: they are active during capacitation by enhanced phosphorylation and, they blocked both protein tyr phosphorylation and the ability of the sperm to acrosome-reaction (Thundathil, De Lamirande and Gagnon, 2002).
Capacitation is also associated with the phosphorylation of MEK and MEK-like proteins
(O’Flaherty, de Lamirande and Gagnon, 2005). The addition of H2O2 increase the levels
of P-MEK-like proteins during capacitation and MEK inhibitors, like PD98059 and U126, are responsible for blocking the rise in P-MEK-like proteins and sperm capacitation (O’Flaherty, de Lamirande and Gagnon, 2006). The capacitation-related increase in P-MEK-like proteins induced by FCSu is modulated by PKA and PKC and it was hypothesized that these P-MEK-like proteins probably represent an intermediary step
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between the early events and the late PKA-dependent Tyr phosphorylation associated with capacitation (O'flaherty, de Lamirande and Gagnon, 2006). Therefore, ROS seem to regulate many of the events related to sperm capacitation.
Figure 6 - Schematic representation of ROS/MAPK pathway in sperm. ROS activate sAC. sAC
promotes the activation of the PKA that activate MEK. H2o2 activates PKC which in turn activates Raf.
PI3K phosphorylates PDK1 which in turn, phosphorylates AKT. AKT stimulates NOsynthesis activating Ras. ROS: Reactive oxygen species; sAC: soluble adenylyl cyclase; PKA: protein kinase A; MEK: mitogen activated kinase; H2o2: hydrogen peroxide; PKC: protein kinase C; Shc: Src homology
collagen; Grb2: Growth factor receptor-bound protein 2; Sos: son of sevenless; ERK: extracellelar signal-regulated kinases; Raf: rapidly accelerated fibrosarcoma; PI3K: phosphatidylinositide 3-kinase; PDK1: phosphoinositide-dependent kinase-1; AKT: protein kinase B; NOS: nitric oxide synthase; NO: nitric oxide; Ras: rat sarcoma protein. cAMP: cyclic adenosine monophosphate. Adapted from O'flaherty, de Lamirande and Gagnon, 2006; Fardilha, Silva and Conde, 2015.
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1.6. Infertility
According to the World Health Organization, infertility is the inability to achieve a pregnancy after twelve months or more keeping unprotected regular intercourse (Zegers-Hochschild et al., 2006). About 25% of couples in reproductive age have fertility-related problems, with an estimated 15% seeking medical treatment for infertility (Kassebaum et al., 2014).
The male factor is involved in 50% of the cases of conjugal infertility, being exclusively responsible for approximately 20% of the cases (Zegers-Hochschild et al., 2009). This percentage has been rising in recent years due to causes as diverse as the postponement of maternity, increased prevalence of sexually transmitted infections, sedentary lifestyle, obesity, tobacco and alcohol consumption and pollution (Jungwirth et al., 2012; Jefferys, Siassakos and Wardle, 2012).
The evaluation of the infertile man should be performed in stages, beginning with the clinical history, physical examination, the cytochemical study of sperm and careful laboratory tests, in the attempt to classify the cause of male infertility.
In Portugal, primary infertility is about 2,1% and secondary infertility 9,2% (Mascarenhas et al, 2012). In Portugal, women age at the first child is increasing: in 1960 the mean age was 25,0 years comparably to 2016 the mean age was 30,3 years (Pordata, 2016). In 2014, a fertility survey performed a Portuguese woman with ages between 18 and 49 years and Portuguese men with ages between 18 and 54 years, showed that the financial costs associated with children and the difficulty in finding a job are the main reasons to postpone paternity (INE, 2014). In other survey, it was concluded that men with highest academic qualifications postponed further paternity in comparison to men with lower qualification. In addition, men (53%) consider having children more important for their personal fulfilment in comparison to women (46%) (FFMS, 2016).
1.6.1. Impact of age on male infertility
Most of age-related fertility studies focus on female age. However, recent studies have shown that advancing male age is associated with changes in semen parameters, compromising fertility (Harris et al., 2011; Sloter et al., 2006).
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The cause of the age-related decline in semen parameters quality has not been clearly defined, however, there are latent age-related diseases, like obesity and genital tract infections, that increase, for instance, the incidence of low progressively motile spermatozoa (Hammoud et al., 2008). The significance of the observed age-related changes in semen parameters remains a controversy because some studies cannot account the relationship between paternal age and reduced pregnancy rate (Koh et al., 2013; Belloc et al., 2014; Keel, 2006).
Several studies indicate that increased male age is associated with a decline in semen volume, sperm motility, and normal sperm morphology but not with sperm concentration (Kidd, Eskenazi and Wyrobek, 2001; Mukhopadhyay et al., 2010).
In contrast, some studies detected that older men have also a significant decrease in sperm concentration (Maya, Berdugo and Jaramillo, 2009), alpha-glucosidase and fructose seminal levels (Molina et al., 2010), and an increase of ROS levels compared with younger men (Cocuzza et al., 2008).
There is also a relationship between sperm DNA quality and age: several studies demonstrated that DNA fragmentation increasing with age, probably caused by defective sperm chromatin packaging, disordered apoptosis and oxidative stress (Agarwal and Said, 2003; Moskovtsev, Willis and Mullen, 2006; Brahem et al., 2011; Evenson et al., 2014). As well, in recent study oxidative stress markers in human semen
were evaluated(Koh, Sanders and Burton, 2016).It was demonstrated that older males
(40 years of age or above) exhibited higher levels of oxidative DNA damage, 8-hydroxy-2′-deoxyguanosine (8-OHdG) and a decrease in sperm concentration and motility. This study suggests that 8-OHdG may be an important biomarker between male age and fertility.
Additionally, advanced paternal age has been associated with lower pregnancy rates, higher risk of pregnancy loss and with childhood health, particularly with higher incidence of congenital birth defects and disorders like achondroplasia, autism, schizophrenia, trisomy and some types of cancers (Sharma et al., 2015).
All these studies showed that the age of male partner has a deleterious impact sperm parameters and DNA quality, which may, in part, contribute to negative reproductive outcomes.
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1.7.
ObjectiveThe studies available regarding the effects of age on male fertility are scarce. Most of those studies are only focused on basic semen parameters and reproductive outcomes. Despite the scientific consensus that male age is an important factor, very little is understood about the molecular mechanisms underlying the relationships between male age and reduced fertility. The main objective of this study is to evaluate the impact of aging on human spermatozoa signaling proteins.
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2.
Material and Methods
2.1. Study Overview
Experimental procedures were performed in Signal Transduction Laboratory, iBiMED Institute for Biomedicine, University of Aveiro (Aveiro, Portugal) and in Unidade de Procriação Medicamente Assistida, COGE – Clínica Obstétrica e Ginecológica de Espinho (Espinho, Portugal).
2.2. Human samples collection
In this study 31 men undergoing Artificial Insemination (AI), In Vitro Fertilization and Intracytoplasmic Sperm Injection (ICSI) treatments or semen analysis at COGE (Clínica Obstétrica e Ginecológica de Espinho) were included. All men signed a written consent authorizing the use of the samples for research purposes (Appendix 1).
2.3. Basic Semen Analysis
Ejaculated human semen samples from donors were collected by masturbation into a sterile container at COGE. Basic semen analysis was conducted in accordance with World Health Organization (WHO, 2010) guidelines. Basic semen analysis was performed using an Eclipse E400 (Nikon) microscope.
2.3.1. Macroscopy parameters evaluation
At the macroscopic level, the following parameters were evaluated: colour, smell, volume, liquefaction, pH and viscosity. The colour and smell were analysed directly by the observer. The volume was measured directly by aspirating the sample from the container into a graduated pipette or 1ml syringe (samples with a volume less than 1ml). After 60 minutes, liquefaction was analysed and pH was measured using pH paper in the range 6,0 to 10,0. The viscosity of the sample was estimated by gently aspirating it into a wide-bore plastic disposable pipette, allowing the semen to drop by gravity.
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2.3.2. Microscopy parameters evaluation
At the microscopic level, the following parameters were evaluated: motility, concentration, vitality, morphology and the presence of leukocytes.
2.3.2.1. Motility
Sperm motility was measured as soon as possible after liquefaction of the sample using Nikon eclipse E400 microscope. Firstly, was prepared a slide with 20ul of fresh sample and using a manual cell counter, two counts (5 different fields) with more than 100 spermatozoa were performed and sperm classified in 4 categories (type a, b, c (in situ) and d).
2.3.2.2. Concentration
For the evaluation of the concentration, a preliminary observation was performed on a slide and this will allow calculating which dilution more suitable to use. Generally, 1:20 dilution (20 ul of semen with 380ul of distilled water) was used. After this, 10ul of the diluted sample was placed in Neubauer hemocytometer (two separate counting chambers) and waited 15 minutes, to allow sedimentation of the spermatozoa. Using Nikon eclipse E400 microscope, the spermatozoa with head and tail of 5 squares (central, upper left, upper right, lower left and lower right) was counted.
2.3.2.3. Vitality
For vitality test, firstly was prepared a 1:1 diluted sample (40ul eosin + 40ul semen). Using Nikon eclipse E400 microscope (phase contrast) was performed 100 counts of spermatozoa with different colours of cell membrane: red/pink (spermatozoa with damaged cell membrane) and greenish (spermatozoa with integrate cell membrane), a total of 100 spermatozoa.
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2.3.2.4. Morphology
To determinate the sperm morphology was prepared a smear of semen on two slides. After air-drying, the slides were fixed and stained (Papanicolaou and Shorr staining). After that, the slides were examined with bright field optics at 1000x magnification with oil immersion, using Nikon eclipse E400. Approximately 200 spermatozoa were assessed and classified, according to the strict criteria of Kruger. Using a cell manual counter were categorized and counted normal spermatozoa, spermatozoa with head defects, spermatozoa with neck and midpiece defects, spermatozoa with tail defects and spermatozoa with excess residual cytoplasm.
2.3.2.5. Leukocytes
The leukocytes were evaluated in a semen smear fixed and stained with the Papanicolaou and Shorr procedures. Using Nikon eclipse E400, the slides were observed with bright field optics at 1000x magnification with oil immersion. All classes of human leukocytes express a specific antigen (CD45) that can be detected with an appropriate monoclonal antibody, to allow detection of different types of leukocytes, such as macrophages, monocytes, neutrophils, B-cells or T-cells.
2.3.3. Semen Processing – Density gradients
The ejaculate contains a mixture of motile, immobile, dead, and possibly agglutinated spermatozoa, in addition to a set of debris (germ cells, exfoliated cells of the male tract, leukocytes and other amorphous material) along with the seminal fluid. Seminal fluid may be toxic to spermatozoa if their contact is delayed, so treatment should be performed poorly if the sperm is liquefied. Density gradients technique is based on the different density presented by spermatozoa. The gradients establish substrates of different densities so that the denser spermatozoa (which are the ones with the best morphology and motility) will go to the bottom of the tube after centrifugation, forming a pellet. This technique is used for Assisted Reproduction Technology (ART).
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2.3.3.1. Semen Preparation by density gradients
After liquefaction, the semen was centrifuged (Labofuge 400, Heraeus) with gradients SupraSperm® (Origio, Denmark) at 55% and 80%, for 20 minutes at 300G. The supernatant was carefully aspirated and the sperm pellet resuspended and washed with Sperm Preparation Medium® (Origio, Denmark) for 10 minutes at 300G. The supernatant was again removed and Sperm Preparation Medium® (Origio, Denmark) was carefully added above the pellet and incubated at 37% at an angle of 45º (Swim-up technique).
2.4. Semen Cryopreservation
After Swim-up technique, Sperm Freezing Medium® (Origio, Denmark) was added by dropwise to the sample in 1:1 ratio. After 10-20 minutes at room temperature, the samples were carefully mixed and placed in horizontal position in liquid nitrogen vapor. After 20-30 minutes, the samples were emerged in liquid nitrogen (-196 ºC) and stored in an appropriate canister.
2.5. Spermatozoa Protein Extracts
Spermatozoa cells were incubated with 1X PathScan Sandwich ELISA Lysis Buffer (Cell Signaling Technology, Danvers, MA, USA) supplemented with 1 mM PMSF for 5 minutes on ice. Then the lysed cells were centrifuged at 14000 g at 4ºC for 10 minutes and the supernatant was transferred to a new tube (cell lysate).
2.6. Protein Quantification - Bicinchoninic Acid Assay
Extracts were mass normalized using the bicinchoninic acid (BCA) assay (Fisher Scientific, Loures, Portugal). Sample was prepared to be assayed with 3 uL of each sample plus 22 uL of the lysis buffer. Standard protein concentrations were prepared as described in Table 1. Samples and standards were prepared in duplicate. The bovine serum albumin (BSA) stock solution used had a concentration of 2 mg/ml. The Working Reagent (WR) was prepared by mixing BCA reagent A with BCA reagent B in the proportion of 50:1.
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Then, 200 µl of WR was added to each well (standards and samples) and the plate was incubated at 37 ºC for 30 min. Once the tubes cooled to RT the absorbance was measured at 562 nm using an Infinite® 200 PRO (Tecan, Switzerland). A standard curve was obtained by plotting BSA standard absorbance vs BSA concentration, and used to determine the total protein concentration of each sample.
Table 1 - Standards for BCA assay.
Standards BSA (µl) 1% SDS (µl) Protein (µg)
P0 - 25 0 P1 0.5 24.5 1 P2 1 24 2 P3 2.5 22.5 5 P4 5 20 10 P5 10 15 20
2.7. Antibody Array - PathScan® Intracellular Signaling Array
Antibody-based arrays were carried out using the PathScan® Intracellular Signaling Array Kit (#7744, Cell Signaling Technology, Danvers, MA, USA) to determine the expression patterns of 18 well-characterized signaling molecules when phosphorylated or cleaved, in 31 semen samples obtained from a randomized group of donors. Each cell extract was diluted to 0,2 ug/ul and applied to its own multiplexed array according to the manufacturer’s instructions. Fluorescence readouts from the arrays were captured digitally using LI-COR® Biosciences Odyssey® imaging system (LI-COR® Biosciences, Nebraska, USA). Pixel intensity was quantified using Odyssey software. The intensity from the negative control within each array was subtracted from all signals, and all data from each array were normalized to the internal positive control within each array.
2.8. Statistical analysis
Statistical analysis was conducted using the IBM SPSS Statistics Software 22. First, a descriptive analysis to each quantitative parameter analyzed was performed. Then, the Pearson correlation coefficient, r, or the Spearman’s rho correlation coefficient, rs, (a nonparametric correlation method) were analyzed to determine the relationship between variables. The significance level was set at 0,05.
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3.
Results
The study included 31 semen samples, obtained from male patients that underwent basic semen analyses at COGE, between March and June 2017. From those, 39% (12 patients) were submitted to ART, namely AI (4 patients), IVF (7 patients) and ICSI (1 patient). The cycles were performed with ejaculated spermatozoa. No pregnancies were achieved. The mean age of men included in this study was 36,8±6,9(SD) years. Data concerning smoking habits was collected (Table 2). From the 31 men included in this study, 5 were smokers. Given the reduced number of smokers in the study sample no further analysis were performed with this parameter.
According to the WHO’s guidelines, basic semen parameters were analyzed and are presented in Table 2. The levels of signaling molecules (in distinct activation states) were determined with the PathScan Intracellular Signaling Array®, which includes antibodies for phosphorylated or cleaved signaling proteins (Table 3).
Initially, to evaluate the relationship between the results obtained from the seminal parameters analysis and patients’ age, a Spearman’s correlation test was performed (Table 4, Appendix 2). Next, the association between the expression patterns of the 18 signaling molecules and age was evaluated using Pearson correlation tests (Table 5). The results indicated that only two basic semen parameters were significantly correlated with age – midpiece defects and excess of residual cytoplasm. Concerning sperm morphology, the percentage of midpiece defects showed a negative correlation with age (Spearman correlation coefficient=-0.43; p=0.012) Contrarily, the results showed a positive association between the excess of residual cytoplasm and age (Spearman correlation coefficient=0.37; p=0.034) (Table 4).
Furthermore, the results indicated that the levels of two phosphoproteins, p70 S6 kinase
(Thr389) (Pearson correlation coefficient= -0,37; p= 0,047) and PRAS40 (Thr246)
(Pearson correlation coefficient=0,54; p=0,002) showed a negative correlation with age (Table 5 and Figure 7).
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Table 2 - Basic semen parameters of 31 patients providing semen samples for ART treatments or semen analysis. FIV, Fertilization In-Vitro; AI, Artificial Insemination;
ICSI, Intracytoplasmic Sperm Injection.
C O D E Techni c A ge (y ears ) Sm ok in g ha bi ts Se xual a bs ti ne nc e (day s) V ol um e ( m L) pH V is cos it y C once nt rat ion (x 10^ 6/ m L) No. Sp e rm at oz oa (x 10^ 6) Im m ot ile (%) Non -p rogr e ss iv e (type c) (%) P rog re ss iv e (t ype b ) (%) Fast pr o gr e ss iv e (type a) (%) V it al it y (%) Norm al m orp hol ogy (%) H e ad de fe ct s ( %) M id pi e ce de fect s (%) Ta il de fects (% ) ERC (%) Le ukocyte s (x 10^ 6/ m L) TZI 25994 Spermogram 29 No 3 2,1 8,1 High 72 151,2 129,2 12,0 25,6 15,4 54 0 98 39 19 11 72 1,67 25204 FIV 36 No 2 3,9 8,1 Normal 60 234,0 185,7 10,3 35,7 22,2 67 5 95 16 2 2 120 1,15 V1000 Spermogram 26 No 3 6,1 8,5 High 145 884,5 89,6 0,4 3,3 6,7 80 9 91 13 5 3 0 1,12 26052 Spermogram 43 No 3 3,5 8,3 High 15 52,5 58,0 30,9 8,8 2,2 33 0 99 20 7 8 30 1,34 25646 FIV 46 No 3 5,0 7,9 Normal 212 1060,0 91,9 0,7 3,0 4,3 73 4 96 13 6 6 0 1,21 25796 Spermogram 39 No 3 8,0 8,1 Normal 33 264,0 70,6 2,7 20,1 6,7 71 5 94 23 2 3 0 1,22 24882 AI 37 No 3 3,4 8,7 Normal 57 193,8 71,3 7,7 17,7 3,3 65 4 96 7 7 2 0 1,12 19108 Spermogram 18 No 3 0,5 8,7 High 40 20,0 19,4 29,1 44,7 6,8 57 5 95 33 4 4 0 1,36 24813 FIV 38 No 3 4,5 8,3 High 92 414,0 84,3 2,6 8,4 4,7 61 5 95 10 13 8 0 1,26 25586 FIV 38 No 3 1,6 8,1 High 114 182,4 71,4 7,4 7,4 13,7 66 5 95 24 1 0 0 1,20 V1100 Spermogram 31 Yes (12 p/day) 5 1,3 8,3 Lower 147 191,1 66,1 4,8 17,6 11,4 66 14 81 10 13 5 0 1,09 26283 Spermogram 33 No 6 6,6 8,1 Normal 113 745,8 90,3 2,3 6,5 0,8 66 3 97 17 3 1 113 1,18 25885 AI 46 No 3 4,3 8,3 Normal 103 442,9 83,9 2,3 9,9 4,0 63 13 88 11 10 3 103 1,12 26342 Spermogram 40 No 3 4,1 8,5 Normal 61 250,1 73,1 5,0 19,3 2,6 73 2 98 23 5 3 61 1,29 26382 Spermogram 38 No 2 4,1 8,1 Lower 86 352,6 80,2 1,8 13,6 4,3 69 17 83 20 3 0 0 1,06 V1200 Spermogram 29 Yes (16 p/day) 3 2,5 9,0 Normal 93 232,5 78,4 3,7 2,7 15,2 60 10 90 33 8 3 0 1,34 26357 ICSI 45 No 3 3,5 8,5 Normal 22 77,0 48,4 11,3 28,3 11,9 62 2 98 14 5 19 0 1,36 26511 Spermogram 44 Yes
(e-cigarette) 2 1,3 8,3 Normal 97 126,1 62,4 7,4 8,9 21,3 68 9 90 12 5 4 0 1,11
26457 Spermogram 36 No 3/4 2,6 8,1 Normal 115 299,0 81,0 4,9 7,3 6,8 59 9 91 29 6 3 230 1,29 24832 AI 34 No 3 1,5 8,7 High 105 157,5 66,6 3,4 18,6 11,4 65 5 95 18 2 1 0 1,16 26531 Spermogram 45 No 5 4,3 7,9 Normal 93 399,9 80,0 1,8 9,4 8,8 79 18 68 28 4 4 0 1,04
University of Aveiro 29 C O D E Techni c A ge (y ears ) Sm ok in g ha bi ts Se xual a bs ti ne nc e (day s) V ol um e ( m L) pH V is cos it y C once nt rat ion (x 10^ 6/ m L) No. Sp e rm at oz oa (x 10^ 6) Im m ot ile (%) Non -p rogr e ss iv e (type c) (%) P rog re ss iv e (t ype b ) (%) Fast pr o gr e ss iv e (type a) (%) V it al it y (%) Norm al m orp hol ogy (%) H e ad de fe ct s ( %) M id pi e ce de fect s (%) Ta il de fects (% ) ERC (%) Le ukocyte s (x 10^ 6/ m L) TZI 26432 Spermogram 32 No 3 3,1 8,1 High 114 353,4 82,3 2,1 10,7 4,9 62 4 94 40 6 1 0 1,41 26493 Spermogram 40 No 4 4,8 7,9 Normal 110 528,0 87,7 1,3 8,8 2,2 61 10 90 34 10 7 110 1,41 26619 Spermogram 42 No 4 0,7 9,0 High 4 2,8 5,6 48,2 44,2 2,0 30 2 98 28 3 5 0 1,34 25823 Spermogram 30 No 3 2,2 8,1 High 35 77,0 56,6 19,1 14,0 10,3 51 3 97 36 5 5 114 1,43 23408 FIV 35 No 3 4,4 8,1 High 55 242,0 69,9 3,5 14,5 12,1 75 5 95 32 2 2 0 1,31 26846 Spermogram 44 No 4 1,9 8,1 Normal 57 108,3 59,4 13,7 24,7 2,2 54 4 96 10 5 5 228 1,16 12520 FIV 46 No 3 1,2 8,1 Normal 92 110,4 56,8 3,1 23,7 16,5 68 8 92 8 3 6 0 1,09 19317 FIV 41 Yes (10 p/day) 3 4,4 8,5 Normal 49 215,6 75,2 3,8 17,8 3,1 60 7 91 17 3 1 4,4 1,12 V1234 Spermogram 24 Yes (15 p/day) 1 6,0 8,3 Lower 27 162,0 63,8 7,1 25,6 3,5 83 8 92 11 3 3 0 1,09 23407 AI 35 No 3 5,2 8,1 High 62 322,4 80,1 1,2 12,4 6,2 65 9 90 28 4 11 0 1,33
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Table 3 - Expression patterns of 18 signaling molecules when phosphorylated or cleaved (PathScan® Intracellular Signaling Array) of 31 patients providing semen
samples for ART treatments or semen analysis.
CODE ERK1/2 (T202/Y204) Stat1 (Y701) Stat3 (Y705) Akt (T308) Akt (S473) AMPKa (T172) S6 Ribossomal Protein (S235/236) mTOR (S2448) HSP27 (S78) Bad (S112) p70 S6 kinase (T389) PRAS40 (T246) p53 (S15) p38 (T180/Y18) SAPK/JNK (T183/Y185) PARP Asp214 Cleavage Caspase-3 Asp175 Cleavage GSK-3b (S9) 12520 0,128 0,138 0,318 0,103 0,118 0,328 0,068 0,138 0,093 0,123 0,113 0,083 0,053 0,068 0,128 0,083 0,133 0,303 19317 0,117 0,117 0,177 0,082 0,127 0,187 0,062 0,127 0,142 0,262 0,067 0,122 0,107 0,067 0,137 0,067 0,177 0,237 25204 0,055 0,065 0,115 0,035 0,055 0,120 0,020 0,050 0,040 0,060 0,040 0,035 0,020 0,030 0,055 0,030 0,050 0,130 24813 0,085 0,090 0,150 0,055 0,085 0,105 0,050 0,060 0,045 0,100 0,095 0,060 0,050 0,050 0,095 0,060 0,070 0,140 25646 0,122 0,152 0,162 0,077 0,092 0,167 0,057 0,087 0,062 0,107 0,092 0,062 0,047 0,047 0,122 0,052 0,077 0,207 25796 0,093 0,093 0,193 0,063 0,083 0,143 0,043 0,063 0,053 0,088 0,098 0,048 0,033 0,038 0,103 0,048 0,068 0,178 25885 0,125 0,105 0,170 0,070 0,100 0,145 0,050 0,080 0,050 0,100 0,090 0,055 0,050 0,055 0,105 0,070 0,075 0,210 26052 0,083 0,068 0,108 0,083 0,063 0,043 0,053 0,073 0,023 0,078 0,088 0,028 0,013 0,058 0,048 0,018 0,073 0,098 26342 0,077 0,077 0,157 0,047 0,062 0,122 0,032 0,057 0,037 0,087 0,052 0,042 0,032 0,037 0,072 0,042 0,057 0,147 26357 0,087 0,092 0,152 0,057 0,077 0,127 0,047 0,082 0,057 0,092 0,077 0,047 0,047 0,057 0,087 0,052 0,077 0,137 26493 0,100 0,080 0,185 0,065 0,075 0,205 0,055 0,060 0,040 0,090 0,100 0,050 0,040 0,050 0,085 0,055 0,070 0,215 26511 0,097 0,117 0,227 0,087 0,092 0,217 0,072 0,097 0,072 0,092 0,087 0,062 0,052 0,047 0,102 0,062 0,092 0,197 26531 0,115 0,115 0,255 0,080 0,120 0,225 0,060 0,110 0,065 0,100 0,065 0,055 0,040 0,065 0,085 0,055 0,080 0,180 26619 0,065 0,055 0,095 0,045 0,055 0,050 0,030 0,050 0,040 0,075 0,075 0,040 0,025 0,035 0,065 0,030 0,055 0,120 26846 0,075 0,090 0,145 0,055 0,070 0,140 0,030 0,100 0,090 0,100 0,045 0,045 0,025 0,035 0,070 0,045 0,130 0,170 V1234 0,145 0,120 0,280 0,105 0,165 0,250 0,075 0,130 0,080 0,130 0,150 0,110 0,070 0,075 0,160 0,105 0,145 0,285 V1200 0,125 0,105 0,300 0,090 0,100 0,270 0,055 0,105 0,090 0,110 0,105 0,090 0,050 0,060 0,130 0,085 0,100 0,210 V1100 0,120 0,110 0,290 0,100 0,100 0,285 0,065 0,135 0,090 0,145 0,075 0,085 0,050 0,040 0,110 0,075 0,135 0,255 V1000 0,125 0,080 0,235 0,065 0,080 0,250 0,040 0,075 0,070 0,085 0,060 0,085 0,035 0,040 0,110 0,055 0,075 0,235 26457 0,110 0,090 0,300 0,080 0,090 0,270 0,050 0,090 0,070 0,090 0,085 0,070 0,045 0,050 0,105 0,070 0,085 0,170 26432 0,103 0,088 0,143 0,093 0,073 0,153 0,078 0,053 0,068 0,073 0,078 0,078 0,038 0,033 0,088 0,048 0,058 0,143 26382 0,117 0,087 0,302 0,072 0,092 0,237 0,052 0,102 0,067 0,097 0,082 0,067 0,052 0,047 0,097 0,067 0,087 0,177 26283 0,112 0,077 0,152 0,067 0,072 0,157 0,042 0,057 0,042 0,057 0,082 0,072 0,027 0,037 0,092 0,037 0,067 0,177 25994 0,065 0,065 0,140 0,080 0,060 0,120 0,030 0,070 0,050 0,070 0,075 0,065 0,030 0,030 0,070 0,045 0,070 0,135
University of Aveiro 31 CODE ERK1/2 (T202/Y204) Stat1 (Y701) Stat3 (Y705) Akt (T308) Akt (S473) AMPKa (T172) S6 Ribossomal Protein (S235/236) mTOR (S2448) HSP27 (S78) Bad (S112) p70 S6 kinase (T389) PRAS40 (T246) p53 (S15) p38 (T180/Y18) SAPK/JNK (T183/Y185) PARP Asp214 Cleavage Caspase-3 Asp175 Cleavage GSK-3b (S9) 25823 0,085 0,060 0,110 0,050 0,060 0,070 0,030 0,040 0,040 0,060 0,075 0,060 0,030 0,030 0,065 0,035 0,040 0,110 25586 0,090 0,065 0,145 0,050 0,070 0,150 0,035 0,050 0,030 0,060 0,070 0,050 0,030 0,030 0,075 0,040 0,055 0,175 24882 0,105 0,080 0,125 0,055 0,080 0,095 0,040 0,060 0,040 0,080 0,095 0,065 0,030 0,040 0,090 0,050 0,065 0,150 24832 0,120 0,090 0,185 0,065 0,085 0,160 0,045 0,070 0,060 0,085 0,115 0,070 0,040 0,050 0,105 0,060 0,070 0,165 23407 0,107 0,102 0,242 0,122 0,117 0,257 0,077 0,132 0,082 0,127 0,117 0,092 0,067 0,072 0,117 0,102 0,142 0,247 23408 0,107 0,107 0,467 0,077 0,097 0,292 0,047 0,122 0,087 0,102 0,067 0,072 0,047 0,057 0,102 0,082 0,107 0,197 19108 0,125 0,080 0,175 0,065 0,085 0,165 0,040 0,070 0,065 0,085 0,125 0,085 0,040 0,050 0,115 0,065 0,080 0,170
University of Aveiro
32
Table 4 - Associations between patients age and the results obtained from the basic seminal
analyses. M Moderate correlation.
Variable Spearman correlation coefficient (p-value) Volume 0.14 (0.4465) pH -0.23 (0.1928) Concentration -0.12 (0.5135) No. spermatozoa 0.02 (0.9220) Immotiled spermatozoa 0.02 (0.9134)
Non-progressive motility (type c) 0.00 (0.9914)
Slow progressive motility (type b) 0.01 (0.9760)
Fast progressive motility (type a) 0.01 (0.9722)
Total spermatozoa motility 0.00 (0.9900)
Progressive spermatozoa motility 0.01 (0.9770)
Vitality -0.05 (0.7857) Normal morphology -0.03 (0.8599) Head defects 0.04 (0.8114) Midpiece defects -0.43 (0.0116) M Tail defects 0.08 (0.6756) Residual cytoplasm 0.37 (0.0343) M TZI -0.23 (0.2031) Leukocytes -0.04 (0.8268)
